Nuclear receptors have become of interest as important key substances for drug discovery. Specifically, screening studies using nuclear receptors have elucidated that tamoxifen, which exhibits an antagonistic effect on an estrogen receptor, is effective as a breast cancer therapeutic agent. However, tamoxifen has been elucidated to exacerbate uterine cancer. It has been suggested that such different effects of a drug which acts on a single nuclear receptor are strongly associated with cofactors of the nuclear receptor.
Conventionally, screening methods for drug discovery using nuclear receptors as key substances have generally employed a receptor binding assay which detects binding of a ligand to a receptor. The detection principle of such a receptor binding assay generally involves, as an index, competitive inhibition of hormone receptor binding of a labeled hormone and a target chemical substance having affinity to the hormone receptor. This method is advantageous for rapid screening of numerous drug candidates. However, although this method can determine the degree of binding of a chemical substance to a receptor, the method encounters difficulty in determining physiological effects of the chemical substance on the receptor; for example, whether the chemical substance exhibits an agonistic effect or an antagonistic effect on the receptor.
Recent studies on such receptors have elucidated that physiological effects of a ligand are strongly dictated by cofactors. Under such circumstances, there has been recognized the utility of a screening system employing cells, such as a reporter gene assay or a two-hybrid assay. However, such an assay, which employs cells, involves problems in that, for example, a culturing apparatus is required; cell culturing requires an intricate process; cell manipulation requires skill to some extent; and a long period of time is required for the assay since cell growth is a rate-determining step in the assay. Therefore, such an assay may be inappropriate as a screening technique for drug discovery requiring rapid screening of numerous samples.
In view of the foregoing, an important key for drug discovery and development is establishment of an in vitro screening system capable of rapidly and conveniently assaying numerous samples on the basis of their mechanisms of physiological action.
Hitherto, forty-eight human nuclear receptors have been known. These include many receptors whose functions have not yet been elucidated (so-called orphan nuclear receptors), and they are considered to play important biological roles. As has been found, several cofactors which act, in conjunction with a receptor, on expression of a specific gene are common to the receptor irrespective of the type of the receptor. As has also been found, the receptor binds to a different type of cofactor depending on the type of a ligand (i.e., an agonist, an antagonist, or a modulator), and the cofactor is involved in expression of a specific gene. When these findings are put into perspective where physiological action of orphan receptors and the relation between cofactors-receptors interaction (combination) and physiological functions (pathological conditions) have been scientifically elucidated, a crucial point in the development of a screening method useful for drug discovery employing a nuclear receptor as a key substance is to establish a high-throughput screening system for drug discovery as soon as possible. Such a screening system must take into consideration contribution of a plurality of different cofactors.
In general, a conventional cofactor-containing receptor-ligand assay system employs a technique similar to that employed in enzyme-linked immunosorbent assay (hereinafter abbreviated as “ELISA”), which is an assay system using an antibody. Conceivably, such a conventional receptor-ligand assay system is established in a manner similar to that in the case of ELISA. Establishment of an assay system based on ELISA requires a long period of time for preparation of a necessary anti-receptor antibody, and also requires selection of assay conditions; for example, selection of conditions for immobilization on a microwell plate, or selection of detection means (use of the fluorescence method or a radioactive substance). Since a variety of nuclear receptor-cofactor combinations are provided, when an assay system is established for each of the combinations as in the case of the aforementioned conventional assay system, a large number of assay systems are required, which is not efficient.
Therefore, an object of the present invention is to provide means for detecting a living-body-related substance, which means employs a nuclear receptor-cofactor system, exhibits high detection sensitivity, provides a convenient detection process, and realizes efficient establishment of a detection system.